The molecular mechanisms that are responsible for the expression and regulation of vaccinia virus (VV) late genes remain an enigma. This class of VV genes, which represents about half of the VV genetic potential, is expressed only after viral DNA synthesis has begun. VV late transcripts from any single locus are highly heterogeneous in size, apparently initiating from a number of distinct 5' sites and terminating randomly. The few VV late genes that have been mapped and sequenced to date, have revealed the conspicious absence of regulatory elements previously recognized as essential in other eukaryotic or prokaryotic systems, or even VV early genes. Thus, the identity of the VV late gene control regions and the viral proteins which presumably recognize them are unknown. Due to the complexity of the situation, one approach to unraveling these questions is to select a few VV late genes of interest, and to subject them to intense molecular biological scrutiny. Towards this end, a number of VV genes which participate in the late phase of VV replication [Alpha-amanitin resistance, ts17, and a tandem array of six non-coordinately expressed VV late genes] have recently been identified, mapped, and sequenced. The experiments seek to use the information obtained thus far as a basis for: 1) A comparative kinetic analysis of how and when these genes are expressed. 2) Preparation of immunological reagents sufficient to enable functional identification of the encoded gene products and how they participate in the viral life cycle. 3) Using directed-genetics, gene fusion, and marker rescue techniques to reveal the salient regulatory features of each gene. 4) Using footprinting and gel retardation methodologies to identify the viral and/or cellular proteins which interact with VV promoter and terminator regions. It is anticipated that such experiments will provide considerable insight into the mechanisms which VV employes to achieve the ordered expression of its complex developmental program within the infected cell. This information may facilitate the design and construction of future VV recombinant vaccine strains to be used for the prophylaxsis of infectious diseases.